Project Summary: Molecular Mechanisms of DNA mismatch repair initiation DNA mismatch repair (MMR) is fundamental to genetic stability. In humans, DNA MMR not only corrects mismatch errors that escape polymerase proofreading in replication, but also it is involved in multiple aspects of cellular physiology including double-strand DNA break repair, recombination, and cellular responses to DNA damage. Failure of DNA MMR in humans is directly linked to several cancers in humans as well as contributing to resistance to chemotherapy. The goal of this project is to determine the molecular interactions that connect DNA mismatch recognition to downstream repair events. How this communication occurs remains one of the most mysterious and controversial aspects of MMR. Multiple populations of the initial mismatch recognition protein complex have been observed ranging from mobile clamps sliding away from the mismatch to static complexes growing at the mismatch. We will use single molecule methods to sort these various subpopulations and determine which interacts with replication processivity clamp, which is essential for subsequent MMR signaling, and will indicate on-repair-pathway states. In our previous publications, we established the existence of these subpopulations (Qiu et al., PNAS 2015). Our project will apply single molecule FRET (smFRET) and tethered particle motion (TPM) experiments to reveal details of the interactions among MMR proteins as well as the impact on DNA conformation. We will develop novel combinations of these assays (TPM+smFRET and smFRET inside live cells) to enable sensitive measurements not previously possible. In addition, we will directly compare results from human and Thermus aquaticus systems to establish conserved features. Guided by strong preliminary data, we designed 3 aims to achieve these goals. Aim 1. Determine MutS:MutL interactions with ?-clamp that drive downstream MMR We will use our smFRET assay to determine whether sliding or static Taq MMR complexes interact with ?-clamp, which is the next, essential step in MMR signaling. Aim 2. In vivo determination of MutS and MutL conformational dynamics with smFRET Using single molecule FRET, we will characterize MMR protein conformational dynamics in live cells. Aim 3. Determine which human and Taq MMR complexes activate downstream excision We will use a tethered particle motion (TPM) assay sensitive to DNA bending, DNA compaction and single strand excision to detect which interactions permit continuation of the MMR cascade beyond the initial recognition complex, to the point of DNA excision. These studies will reveal the basic mechanisms that underlie mismatch repair, which will be important for designing treatment of cancers involving malfunction of DNA mismatch repair.